Immunoprecipitation combined with micro capillary gel electrophoresis on chip, gas-phase electrophoretic mobility macromolecular analyzer (GEMMA) and matrix assisted laser desorption ionization mass spectrometry (MALDI MS) of proteins and conjugates

Abstract

Abstract In the first part of the thesis, aim of the work was to combine the concept of antibody affinity with a micro fluidic separation to build finally a complete lab-on-a-chip device. In a first approach immunoprecipitation with different antibodies was used for selective enrichment followed by separation and detection (LIF) of several proteins of interest from different biological sources under various conditions. The off-line immunoprecipitation procedure for different antibody-antigen complexes was developed and optimized for capillary gel electrophoresis-on-a-chip system. Finally, the molecular size and amount of the precipitated biomolecules were determined via capillary gel electrophoresis-on-a-micro fluidic device with LIF detection. As a final perspective, the miniaturization, integration and application of the whole method to a commercial lab-on-a-chip device were discussed and the feasibility of this methodology was demonstrated.<br />In the second part of the work, the exact molecular weight determination of a drug conjugate was investigated. The uptake of the drug into the cells could be enhanced via coupling of the drug to a lectin. In a bioassay-guided development process, the applicability of this novel delivery approach to a protein model-prodrug was evaluated.<br />Fluorescein-labeled bovine serum albumin (fBSA) was covalently coupled to wheat germ agglutinin (WGA). The determination of the exact molecular weight and modification degree of the conjugate could help understanding the relationship between the structure and the binding efficacy of the conjugate to the cells and transfer efficacy of the drug into the cells to further optimize the production of conjugate with the wanted degree of modification for a higher efficiency during drug delivery. For the determination of moleclular weight and modification degree of the produced conjugate, several methods like SDS-PAGE, GEMMA and MALDI MS were performed.